Qhov Tseem Ceeb Qhov txawv - NGS vs Sanger Sequencing
Next Generation Sequencing (NGS) thiab Sanger Sequencing yog ob hom nucleotide sequencing cov tswv yim tsim los ntawm lub sijhawm. Sanger Sequencing txoj kev tau siv dav rau ntau xyoo thiab NGS hloov nws tsis ntev los no vim nws qhov zoo. Qhov sib txawv tseem ceeb ntawm NGS thiab Sanger Sequencing yog tias NGS ua haujlwm ntawm lub hauv paus ntsiab lus ntawm kev sib sau ntau lab ntawm cov kab ke ib txhij nyob rau hauv txoj kev nrawm los ntawm kev sib sau ua ke thaum Sanger Sequencing ua haujlwm ntawm tus thawj coj ntawm cov saw txiav vim xaiv kev koom ua ke ntawm dideoxynucleotides los ntawm DNA polymerase enzyme. Thaum lub sij hawm DNA replication thiab ua rau tawg tawg tawg los ntawm capillary electrophoresis.
Nucleotide Sequencing yog dab tsi?
Cov ntaub ntawv caj ces yog khaws cia hauv nucleotide sequences ntawm DNA lossis RNA ntawm lub cev. Cov txheej txheem ntawm kev txiav txim siab qhov tseeb ntawm nucleotides (siv plaub lub hauv paus) nyob rau hauv ib qho khoom tawg (hauv ib lub noob, pawg ntawm cov noob, chromosome, thiab tiav genome) yog hu ua nucleotide sequencing. Nws yog ib qho tseem ceeb heev nyob rau hauv kev tshawb fawb genomic, kev tshawb fawb forensic, virology, biological systematic, kev kuaj mob, biotechnology thiab nyob rau hauv ntau lwm yam kev soj ntsuam cov qauv thiab kev ua ntawm noob. Muaj ntau hom kev sib txuas ua ke tsim los ntawm cov kws tshawb fawb. Ntawm lawv, Sanger sequencing tsim los ntawm Frederick Sanger hauv xyoo 1977 tau siv dav thiab nrov rau lub sijhawm ntev mus txog Lub Neej Tom Ntej Sequencing hloov nws.
NGS yog dab tsi?
Next Generation Sequencing (NGS) yog ib lo lus siv los xa mus rau cov txheej txheem kev sib tw ua ntu zus niaj hnub no. Nws piav txog ntau yam kev sib txawv niaj hnub sib txawv uas tau hloov pauv kev tshawb fawb genomic thiab Molecular Biology. Cov txheej txheem no yog Illumina sequencing, Roche 454 sequencing, Ion Proton sequencing thiab SOLiD (Sequencing by Oligo Ligation Detection) sequencing. NGS systems yog ceev dua thiab pheej yig dua. Plaub lub ntsiab DNA sequencing txoj kev yog siv nyob rau hauv NGS systems namely; pyrosequencing, sequencing los ntawm synthesis, sequencing los ntawm ligation thiab ion semiconductor sequencing. Ntau tus DNA lossis RNA strands (ntau lab ntawm) tuaj yeem ua ke ua ke. Nws tso cai rau kev sib luag ntawm tag nrho cov genome ntawm cov kab mob hauv lub sijhawm luv luv, tsis zoo li Sanger sequencing uas yuav siv sij hawm ntau dua.
NGS muaj ntau yam zoo dua li cov qauv sib txuas ntawm Sanger. Nws yog cov txheej txheem kev kub ceev, muaj tseeb dua thiab tus nqi tsim nyog uas tuaj yeem ua nrog cov qauv me me. NGS tuaj yeem siv rau hauv cov kev tshawb fawb metagenomic, hauv kev tshawb nrhiav qhov txawv ntawm ib tus neeg genome vim kev ntxig thiab tshem tawm thiab lwm yam.
Figure_1: Kev txhim kho hauv NGS Sequences
Sanger Sequencing yog dab tsi?
Sanger Sequencing yog ib txoj hauv kev tsim los ntawm Frederick Sanger thiab nws cov npoj yaig hauv xyoo 1977 los txiav txim siab qhov tseeb nucleotide kev txiav txim ntawm ib qho DNA tawg. Nws tseem hu ua chain termination sequencing los yog Dideoxy sequencing. Lub hauv paus ntsiab lus ua haujlwm ntawm txoj kev no yog kev txiav tawm ntawm strand synthesis los ntawm kev xaiv kev sib koom ua ke ntawm cov saw hlau txiav tawm dideoxynucleotides (ddNTPs) xws li ddGTP, ddCTP, ddATP thiab ddTTP los ntawm DNA polymerase thaum lub sijhawm rov ua dua ntawm DNA. Cov nucleotides ib txwm muaj 3 'OH pawg rau kev tsim cov phosphodiester daim ntawv cog lus ntawm cov nucleotides uas nyob ib sab mus txuas ntxiv cov strand. Txawm li cas los xij, ddNTPs tsis muaj 3 'OH pawg no thiab tsis tuaj yeem tsim cov phosphodiester bonds ntawm nucleotides. Li no, cov saw elongation yog ceased.
Nyob rau hauv txoj kev no, ib leeg-stranded DNA yuav tsum ua raws li tus qauv strand rau hauv vitro DNA synthesis. Lwm qhov yuav tsum tau muaj yog oligonucleotide primer, deoxynucleotide precursors thiab DNA polymerase enzyme. Thaum lub flanking kawg ntawm lub hom phiaj fragment paub, primers tau yooj yim tsim rau DNA replication. Plaub cais DNA synthesis tshwm sim yog ua nyob rau hauv plaub cais hlab. Txhua lub raj muaj cais ddNTPs, ua ke nrog lwm cov kev cai. Los ntawm cov nucleotide tshwj xeeb, sib xyaw ntawm dNTPs thiab ddNTPs tau ntxiv. Ib yam li ntawd, plaub qhov kev sib cais yog ua nyob rau hauv plaub lub raj nrog plaub qhov sib tov. Tom qab cov tshuaj tiv thaiv, kev tshawb pom ntawm DNA tawg thiab hloov pauv ntawm cov qauv fragment rau hauv cov ntaub ntawv ua ntu zus. Qhov tshwm sim DNA tawg yog kub denatured thiab sib cais los ntawm gel electrophoresis. Yog tias siv cov xov tooj cua nucleotides, cov qauv banding hauv polyacrylamide gel tuaj yeem pom los ntawm autoradiography. Thaum txoj kev no siv cov fluorescently tagged dideoxynucleotides, nws tuaj yeem txo qis hauv cov gel nyeem thiab dhau los ntawm lub teeb ntawm laser kom kuaj tau los ntawm lub ntsuas fluorescent. Txhawm rau zam qhov tsis raug uas yuav tshwm sim thaum ib ntu tau nyeem los ntawm lub qhov muag thiab nkag mus rau hauv lub khoos phis tawj manually, txoj kev no tau tsim los rau hauv kev siv cov khoom sib txuas ua ke nrog lub computer.
Nov yog txoj kev siv los ua kom DNA los ntawm Human Genome project. Txoj kev no tseem siv tau nrog cov kev hloov kho siab heev vim nws muab cov ntaub ntawv qhia meej meej txawm tias yog tus txheej txheem kim thiab qeeb.
Figure_2: Sanger Sequencing
NGS thiab Sanger Sequencing yog dab tsi?
NGS vs Sanger Sequencing |
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| Next Generation Sequencing (NGS) xa mus rau cov txheej txheem kev sib tw ua ntu zus niaj hnub no. Nws piav txog ntau yam kev sib txawv niaj hnub ua ntu zus | Sanger Sequencing yog ib txoj hauv kev tsim los ntawm Frederick Sanger los txiav txim qhov tseeb nucleotide kev txiav txim ntawm cov khoom DNA muab. |
| Nyiaj tau txiaj ntsig | |
| NGS yog cov txheej txheem pheej yig dua vim tias nws txo lub sijhawm, txiv neej lub zog thiab tshuaj. | Qhov no yog txheej txheem kim vim nws siv sijhawm, txiv neej lub zog thiab ntau yam tshuaj. |
| Qhov no sai dua vim tias ob qho tib si tshuaj lom neeg tshawb pom thiab pom cov teeb liab ntawm ntau cov strands tau tshwm sim tib lub sijhawm. | Qhov no yog siv sijhawm ntev txij li kev tshawb nrhiav tshuaj lom neeg thiab kev kuaj pom tshwm sim raws li ob txoj haujlwm sib cais thiab tsuas yog ntawm strand tuaj yeem nyeem ib zaug xwb. |
| NGS yog ntseeg tau. | Sanger sequencing is less ntseeg |
| Sample Size | |
| NGS xav tau DNA tsawg dua. | Txoj kev no xav tau ntau tus qauv DNA. |
| DNA Bases ib ntu ntu | |
| Tus naj npawb ntawm DNA hauv paus ib ntu ntu qis dua Sanger txoj kev | Kev tsim cov kab ke ntev dua li NGS sequences. |
Summary – NGS vs Sanger Sequencing
NGS thiab Sanger Sequencing yog cov txheej txheem nucleotide sequencing siv dav hauv Molecular Biology. Sanger sequencing yog ib txoj hauv kev thaum ntxov uas tau hloov los ntawm NGS. Qhov sib txawv tseem ceeb ntawm NGS thiab Sanger Sequencing yog tias NGS yog cov txheej txheem kev kub ceev, raug thiab raug nqi ntau dua li Sanger sequencing. Ob lub tswv yim tsim kev sib kis loj hauv Genetics thiab Biotechnology.
